Test method for simulating degradation process of starch embolism microspheres in vivo
By using pig pancreatic α-amylase and Tris-HCl buffer in the simulated solution, the simulated solution was replaced every time, the sugar quality changes in starch embolization microspheres were measured, and the degradation rate was calculated, and the starch embolization in the prior art was solved. The problem of inaccurate microsphere degradation process has achieved accurate simulation and material optimization of the degradation process of starch embolized microspheres in vivo.
Patent Information
- Application Number
- CN202411408210.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-08
- Publication Date
- 2025-01-03
AI Technical Summary
The existing research methods for simulating the degradation process of starch embolized microspheres in vivo are not accurate enough. They are mainly analyzed through total volume changes, particle size changes or total mass changes, and cannot accurately simulate the real degradation process of starch embolized microspheres in vivo.
Specific simulation fluids, including pig pancreatic α-amylase and Tris-HCl buffer, simulate the enzyme environment in the plasma in vivo, and replace the simulation fluid at preset times during the incubation process to measure the reduction in starch embolization microspheres. The relationship between sugar mass and time is calculated by formulas to simulate the real degradation process of starch embolized microspheres in the body.
This method can more accurately simulate the degradation process of starch embolized microspheres in the body, provide scientific basis for medical device registration approval and material development, and reduce the risks and costs of clinical trials.
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Figure CN119242767A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of embolic microspheres, and in particular to a testing method for simulating the degradation process of starch embolic microspheres in vivo. Background Art
[0002] Starch embolic microspheres are a biodegradable material that is widely used in the medical field, mainly for hepatic artery embolization and cerebral vascular embolism. The degradation process of starch embolic microspheres in the body (including degradation time, etc.) is one of the important indicators to measure its performance.
[0003] At present, there are relatively few studies on simulating the degradation process of starch embolic microspheres in vivo, which mainly include analyzing the degradation process through the total volume change, particle size change or total mass change of starch embolic microspheres; however, the above methods are quite different from the actual degradation process of starch embolic microspheres in vivo, and cannot accurately simulate the actual degradation process of starch embolic microspheres in vivo. Summary of the invention
[0004] The purpose of the present application is to provide a testing method for simulating the degradation process of starch embolic microspheres in vivo, which can more accurately simulate the actual degradation process of starch embolic microspheres in vivo.
[0005] The present application provides a test method for simulating the degradation process of starch embolic microspheres in vivo, the test method comprising: placing the starch embolic microspheres in a simulated liquid for incubation; and during the incubation process, removing the original simulated liquid at preset intervals and adding new simulated liquid; obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time to determine the degradation process of the starch embolic microspheres in vivo. The simulated liquid contains porcine pancreatic α-amylase and Tris-HCl buffer; the pH of the Tris-HCl buffer is 7.35-7.45; the enzyme activity concentration of porcine pancreatic α-amylase in the simulated liquid is the same as the enzyme activity concentration of α-amylase in the simulated plasma in vivo. The incubation temperature is 36-38°C. The preset time is ≤24h.
[0006] The method provided in the present application uses a specific simulation liquid and replaces the simulation liquid at specific preset time intervals, so as to simulate the real enzymatic degradation environment of starch embolic microspheres in vivo; by obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time, it reflects the change of the degradation of the starch embolic microspheres with the incubation time, and thus can more accurately simulate the real degradation process of the starch embolic microspheres in vivo; the method provided in the present application can be used to evaluate the degradation behavior of starch embolic microspheres in vivo, which can provide a scientific basis for the registration and approval process of medical devices, and also help the development and optimization of starch embolic microsphere materials, reducing the risks and costs of clinical trials.
[0007] In an optional implementation manner of the present application, the preset time is 18 to 24 hours.
[0008] The above technical solution can make the incubation system more accurately simulate the real enzymatic degradation environment of starch embolization microspheres in vivo.
[0009] In an optional embodiment of the present application, the incubation is carried out in a shaker with a rotation speed of 60 to 80 rpm.
[0010] The above technical solution can make the incubation system more accurately simulate the real enzymatic degradation environment of starch embolization microspheres in vivo.
[0011] In an optional embodiment of the present application, the reduced sugar mass M in the starch embolization microspheres is obtained according to formula (1): t The formula (1) is as follows:
[0012] M t =M s +M e
[0013] Where M s is: the total mass of sugar removed from all simulated solutions during the incubation process; M e is: the mass of sugar in the supernatant after centrifugation of the incubated system.
[0014] The above technical scheme can obtain the reduced sugar mass M in the starch embolization microspheres at different incubation times. t , and then obtain the relationship between the reduced sugar mass in the starch embolization microspheres and the incubation time to simulate the degradation process of starch embolization microspheres in vivo.
[0015] In an optional embodiment of the present application, the testing method includes: obtaining the relationship between the degradation rate of starch embolic microspheres and the incubation time based on the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time, so as to determine the degradation process of the starch embolic microspheres in vivo; wherein the degradation rate DR of the starch embolic microspheres is obtained according to formula (2).
[0016] Formula (2) is as follows:
[0017] Where M0 is the mass of sugar in starch plugged microspheres before incubation.
[0018] The above technical solution can simulate the degradation process of starch embolization microspheres in vivo according to the determined relationship between the degradation rate of starch embolization microspheres and the incubation time.
[0019] In an optional embodiment of the present application, the sugar concentration in all the simulated fluids removed is tested to determine the Ms ; Test the sugar concentration in the supernatant to determine M e .
[0020] Optionally, the concentration of sugar in all the simulated liquid removed and the concentration of sugar in the supernatant are tested using anthrone colorimetry.
[0021] In an optional embodiment of the present application, the starch embolic microspheres are pretreated before incubation; the pretreatment step includes: replacing the starch embolic microspheres with physiological saline, collecting the precipitate after centrifugation, and obtaining the starch embolic microspheres from the precipitate.
[0022] In an optional embodiment of the present application, a preset volume of starch embolization microspheres is placed in a simulated liquid for incubation; the volume ratio of the preset volume to the simulated liquid during the incubation process is 1:(20-30).
[0023] The above technical solution can make the incubation system more accurately simulate the real enzymatic degradation environment of starch embolization microspheres in vivo.
[0024] In an optional embodiment of the present application, the starch embolic microspheres have a core-shell structure, and the degradation rate of the core material of the starch embolic microspheres is greater than the degradation rate of the shell material of the starch embolic microspheres.
[0025] Optionally, the core material is a soluble starch polymer, or / and the shell material is a pullulan polymer.
[0026] Optionally, in the starch embolization microspheres, the mass ratio of the core material to the shell material is (1-5):(1-5).
[0027] In an optional embodiment of the present application, the preparation method of starch embolic microspheres includes: preparing a microsphere skeleton material into an aqueous phase, the microsphere skeleton material includes a core material and a shell material, the hydrophilicity of the core material is greater than that of the shell material; the aqueous phase includes sodium trimetaphosphate and sodium tripolyphosphate; the aqueous phase and the oil phase are mixed to form an inverse suspension polymerization system, and then a polymerization reaction is performed to obtain starch embolic microspheres. The test method includes: obtaining the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolic microspheres and the incubation time to determine the degradation process of the starch embolic microspheres in vivo.
[0028] In the above technical scheme, for starch embolic microspheres prepared with phosphorus-containing substances (i.e., sodium trimetaphosphate and sodium tripolyphosphate), by obtaining the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolic microspheres and the incubation time, it is possible to more accurately reflect the change in the degradation of the starch embolic microspheres with the incubation time, and thus more accurately simulate the actual degradation process of the starch embolic microspheres in the body.
[0029] Optionally, the sum of the reduced sugar mass and phosphorus mass in the starch embolization microspheres M is obtained according to formula (3): 总 ; Formula (3) is as follows:
[0030] M 总 =M s +M e +M a +M b
[0031] Where M s is: the total mass of sugar removed from all simulated solutions during the incubation process; M e is: the mass of sugar in the supernatant after centrifugation of the incubated system; M a is: the total mass of phosphorus removed from all simulated solutions during the incubation process; M b is: the mass of phosphorus in the supernatant after centrifugation of the incubated system. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0033] Figure 1 A flow chart of a test method for simulating the degradation process of starch embolization microspheres in vivo provided in the present application.
[0034] Figure 2 It is a normalized curve diagram of incubation time and degradation rate of the embodiment.
[0035] Figure 3 This is the result 2 hours after rabbit ear embolization.
[0036] Figure 4 This is the result 5 days after rabbit ear embolization.
[0037] Figure 5 This is the result of rabbit ear embolization 7 days later.
[0038] Figure 6 This is the degradation diagram of starch embolic microspheres with low cross-linking degree in Comparative Example 1.
[0039] Figure 7 This is the degradation diagram of starch embolic microspheres with high cross-linking degree in Comparative Example 1. DETAILED DESCRIPTION
[0040] The present application provides a test method for simulating the degradation process of starch embolic microspheres in vivo, the test method comprising: placing the starch embolic microspheres in a simulated liquid for incubation; and during the incubation process, removing the original simulated liquid at preset intervals and adding new simulated liquid; obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time to determine the degradation process of the starch embolic microspheres in vivo.
[0041] The simulated liquid contains porcine pancreatic α-amylase and Tris-HCl buffer; the pH of the Tris-HCl buffer is 7.35-7.45; the enzyme activity concentration of the porcine pancreatic α-amylase in the simulated liquid is the same as the enzyme activity concentration of the α-amylase in the simulated plasma in vivo.
[0042] The incubation temperature is 36-38°C.
[0043] The preset time is ≤24h.
[0044] The method provided in the present application uses a specific simulation liquid and replaces the simulation liquid at specific preset time intervals, so as to simulate the real enzymatic degradation environment of starch embolic microspheres in vivo; by obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time, it reflects the change of the degradation of the starch embolic microspheres with the incubation time, and thus can more accurately simulate the real degradation process of the starch embolic microspheres in vivo; the method provided in the present application can be used to evaluate the degradation behavior of starch embolic microspheres in vivo, which can provide a scientific basis for the registration and approval process of medical devices, and also help the development and optimization of starch embolic microsphere materials, reducing the risks and costs of clinical trials.
[0045] It is understandable that if the incubation time is less than the preset time, the simulation liquid does not need to be replaced during the incubation process; the starch embolic microspheres will not be removed during the operation of "removing the original simulation liquid and adding new simulation liquid", and the starch embolic microspheres are always in the incubation system; "simulated in vivo plasma" refers to the in vivo plasma of the simulated object. For example, if the degradation process of starch embolic microspheres in rabbits is to be simulated, the simulated in vivo plasma is rabbit plasma.
[0046] As an example, the pH of the Tris-HCl buffer can be any value among 7.35, 7.37, 7.4, 7.42 and 7.45, or a range between any two of them; the incubation temperature can be any value among 36°C, 36.5°C, 37°C, 37.5°C and 38°C, or a range between any two of them.
[0047] Figure 1 For a flow chart of the test method for simulating the degradation process of starch embolic microspheres in vivo provided in this application, please refer to Figure 1The test method for simulating the degradation process of starch embolic microspheres in vivo includes the following steps:
[0048] S110, placing the starch embolization microspheres in a simulated liquid for incubation; and during the incubation process, removing the original simulated liquid at a preset time interval and adding new simulated liquid.
[0049] In some optional embodiments of the present application, the preset time is 18 to 24 hours; this allows the incubation system to more accurately simulate the actual enzymatic degradation environment of the starch embolization microspheres in vivo.
[0050] As an example, the preset time may be any point value among 18h, 19h, 20h, 21h, 22h, 23h and 24h, or a range value between any two of them.
[0051] In some optional embodiments of the present application, the incubation is carried out in a shaker with a rotation speed of 60 to 80 rpm; this allows the incubation system to more accurately simulate the actual enzymatic degradation environment of the starch embolization microspheres in vivo.
[0052] As an example, the rotation speed of the shaker can be any value among 60 rpm, 65 rpm, 70 rpm, 75 rpm and 80 rpm, or any range between any two values.
[0053] In some optional embodiments of the present application, the starch embolic microspheres are pretreated before incubation; the pretreatment step includes: replacing the starch embolic microspheres with physiological saline, collecting the precipitate after centrifugation, and obtaining the starch embolic microspheres from the precipitate.
[0054] Furthermore, a preset volume of starch embolism microspheres is placed in a simulated liquid for incubation; the volume ratio of the preset volume to the simulated liquid during incubation is 1:(20-30); the incubation system can more accurately simulate the real enzymatic degradation environment of starch embolism microspheres in vivo.
[0055] As an example, the volume ratio of the preset volume to the simulated liquid during the incubation process can be any value among 1:20, 1:22, 1:25, 1:27 and 1:30 or a range between any two values.
[0056] In the present application, the simulated liquid contains porcine pancreatic α-amylase and Tris-HCl buffer; the pH of the Tris-HCl buffer is 7.35-7.45; the enzyme activity concentration of porcine pancreatic α-amylase in the simulated liquid is the same as the enzyme activity concentration of α-amylase in the simulated plasma in vivo.
[0057] As an example, the simulation solution is prepared by mixing porcine pancreatic α-amylase stock solution with Tris-HCl buffer. It should be noted that the porcine pancreatic α-amylase stock solution should be used up within 7 days of preparation to avoid the activity of porcine pancreatic α-amylase being reduced and affecting the test results.
[0058] S120, obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time to determine the degradation process of the starch embolic microspheres in vivo.
[0059] In some optional embodiments of the present application, the reduced sugar mass M in the starch embolization microspheres is obtained according to formula (1): t The formula (1) is as follows:
[0060] M t =M s +M e
[0061] Where M s is: the total mass of sugar removed from all simulated solutions during the incubation process.
[0062] M e is: the mass of sugar in the supernatant after centrifugation of the incubated system.
[0063] Understandably, M t Indicates: when the incubation time is t, the reduced sugar mass in the starch plugged microspheres is obtained; M s It means: when the incubation time is t, the total mass of sugar in all simulated liquids that has been removed from the incubated system; M e It means: when the incubation time is t, the mass of sugar in the supernatant after centrifugation of the incubated system.
[0064] Get M respectively s and M e , and by using formula (1), the reduced sugar mass M in starch plugged microspheres at different incubation times can be obtained t , and then obtain the relationship between the reduced sugar mass in the starch embolization microspheres and the incubation time (ie, degradation time) to simulate the degradation process of starch embolization microspheres in vivo.
[0065] Further, the test method includes: according to the relationship between the reduced sugar mass in the starch embolization microspheres and the incubation time, obtaining the relationship between the degradation rate of the starch embolization microspheres and the incubation time, so as to determine the degradation process of the starch embolization microspheres in vivo; wherein, the degradation rate DR of the starch embolization microspheres is obtained according to formula (2). Formula (2) is as follows:
[0066]
[0067] Where M0 is the mass of sugar in starch plugged microspheres before incubation.
[0068] According to the obtained M t And M0, and according to formula (2), the relationship between the degradation rate of starch embolism microspheres and the incubation time (i.e., degradation time) can be obtained; according to the determined relationship between the degradation rate of starch embolism microspheres and the incubation time (i.e., degradation time), the degradation process of starch embolism microspheres in vivo is simulated.
[0069] In some optional embodiments of the present application, the sugar concentration in all the simulated fluids removed is tested to determine the M s ; Test the sugar concentration in the supernatant to determine M e The corresponding mass can be obtained based on the concentration and volume tested.
[0070] Furthermore, the concentration of sugar in all the removed simulated liquids and the concentration of sugar in the supernatant were tested by anthrone colorimetry.
[0071] As an example, the concentration of glucose in all the simulated fluids removed and the concentration of glucose in the supernatant were tested using anthrone colorimetric method.
[0072] In some optional embodiments of the present application, in order to more accurately simulate the degradation process of starch embolization microspheres in vivo, the degradation rates corresponding to at least five different incubation times may be obtained.
[0073] In some optional embodiments of the present application, the starch embolic microspheres to be tested have a core-shell structure, and the degradation rate of the core material of the starch embolic microspheres is greater than the degradation rate of the shell material of the starch embolic microspheres.
[0074] The starch embolization microspheres with core-shell structure to be tested are described in detail below.
[0075] Furthermore, the core material of the starch embolic microspheres to be tested is a soluble starch polymer; and the shell material of the starch embolic microspheres to be tested is a branched-chain starch polymer.
[0076] Furthermore, in the starch embolization microspheres to be tested, the mass ratio of the core material to the shell material is (1-5):(1-5).
[0077] In some optional embodiments of the present application, the preparation method of the above-mentioned starch embolic microspheres to be tested with a core-shell structure includes: preparing the microsphere skeleton material into an aqueous phase, the microsphere skeleton material includes a core material and a shell material, the degradation rate of the core material is greater than the degradation rate of the shell material, and the hydrophilicity of the core material is greater than the hydrophilicity of the shell material; mixing the aqueous phase and the oil phase to form a reverse suspension polymerization system, and then performing a polymerization reaction to obtain the starch embolic microspheres to be tested.
[0078] The aqueous phase includes microsphere skeleton material, sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STTP); the microsphere skeleton material includes core material and shell material, the core material is a soluble starch polymer, and the shell material is a branched-chain starch polymer.
[0079] Furthermore, during the aqueous phase preparation process, the mass ratio of the soluble starch polymer to the branched-chain starch polymer is (1-5):(1-5), and the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate is (5-10):1.
[0080] The oil phase includes cyclohexane, Span 60 and Tween 60.
[0081] Furthermore, during the reverse polymerization process, the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate is (5-10):1; the mass ratio of Span 60 to Tween 60 is (1-3):(0.5-1.5).
[0082] Furthermore, the polymerization reaction includes a first-stage reaction and a second-stage reaction; the temperature of the first-stage reaction is 60-80°C; the time of the first-stage reaction is 2-4 hours; the temperature of the second-stage reaction is 60-80°C; the time of the second-stage reaction is 18-24 hours.
[0083] Furthermore, sodium trimetaphosphate and sodium tripolyphosphate are added during the second-stage reaction.
[0084] As an example, the method for preparing the above-mentioned starch embolization microspheres with a core-shell structure comprises the following steps:
[0085] Aqueous phase configuration: under stirring conditions, sodium trimetaphosphate and sodium tripolyphosphate are added to the mixed solution of soluble starch polymer and amylopectin polymer to obtain an aqueous phase. Oil phase configuration: under stirring conditions, Span 60 and Tween 60 are added to cyclohexane to obtain an oil phase. Reverse polymerization: while maintaining stirring, add the aqueous phase to the oil phase, control the reaction temperature to 60-80°C, react for 2-4 hours, add sodium trimetaphosphate and sodium tripolyphosphate and continue to react for 18-24 hours to obtain the starch embolization microspheres to be tested.
[0086] For the starch embolic microspheres with a core-shell structure prepared by the phosphorus-containing substances (i.e., sodium trimetaphosphate and sodium tripolyphosphate), the test method for simulating the degradation process of the starch embolic microspheres in vivo includes: obtaining the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolic microspheres and the incubation time to determine the degradation process of the starch embolic microspheres in vivo.
[0087] For starch embolic microspheres prepared with phosphorus-containing substances (i.e., sodium trimetaphosphate and sodium tripolyphosphate), by obtaining the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolic microspheres and the incubation time (i.e., degradation time), it is possible to more accurately reflect the change in the degradation amount of starch embolic microspheres with the degradation time, and thus more accurately simulate the actual degradation process of starch embolic microspheres in vivo.
[0088] Further, according to formula (3), the sum of the reduced sugar mass and phosphorus mass in the starch embolization microspheres M is obtained: 总 ; Formula (3) is as follows:
[0089] M 总 =M s +M e +M a +M b
[0090] Where M s is: the total mass of sugar removed from all simulated solutions during the incubation process.
[0091] M e is: the mass of sugar in the supernatant after centrifugation of the incubated system.
[0092] M a is the total mass of phosphorus removed from all simulated solutions during the incubation process.
[0093] M b is: the mass of phosphorus in the supernatant after centrifugation of the incubated system.
[0094] Understandably, M 总 Indicates: when the incubation time is t, the sum of the reduced sugar mass and phosphorus mass in the starch plugged microspheres is obtained; M s It means: when the incubation time is t, the total mass of sugar in all simulated liquids that has been removed from the incubated system; M e It means: when the incubation time is t, the mass of sugar in the supernatant after centrifugation of the incubated system; M a It means: when the incubation time is t, the total mass of phosphorus in all simulated liquids removed from the incubated system; M b It means: when the incubation time is t, the mass of phosphorus in the supernatant after centrifugation of the incubated system.
[0095] Furthermore, the test method includes: according to the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolization microspheres and the incubation time, obtaining the relationship between the degradation rate of the starch embolization microspheres and the incubation time, so as to determine the degradation process of the starch embolization microspheres in vivo; wherein the degradation rate DR of the starch embolization microspheres is obtained according to formula (4). Formula (4) is as follows:
[0096]
[0097] Wherein, M0' is the sum of the mass of sugar and the mass of phosphorus in the starch plugged microspheres before incubation.
[0098] In some optional embodiments of the present application, the sugar concentration in all the simulated fluids removed is tested to determine the M s ; Test the sugar concentration in the supernatant to determine M e ; Test the phosphorus concentration in all simulated solutions removed to determine the M a ; Test the phosphorus concentration in the supernatant to determine M b .
[0099] As an example, the total phosphate content determination method according to GBT 6913-2008 is used to test the phosphorus concentration, and the anthrone colorimetric method is used to test the sugar concentration.
[0100] As an example, the concentration of glucose in all the simulated liquids removed and the concentration of glucose in the supernatant were tested by anthrone colorimetry, and the concentration of glucose in all the simulated liquids removed and the phosphate (measured as PO4 in the supernatant) were tested by GBT 6913-2008 total phosphate content determination method. 3- The concentration of the
[0101] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0102] Example
[0103] This embodiment provides a test method for simulating the degradation process of starch embolization microspheres in vivo, which comprises the following steps:
[0104] (1) Preparation of starch embolic microspheres:
[0105] Preparation of corn amylopectin solution: Add 10g corn amylopectin and 1g NaOH into 100mL distilled water, stir and heat until gelatinized and transparent, let stand and cool for use.
[0106] Preparation of soluble starch solution: Add 10 g soluble starch and 1 g NaOH into 100 mL distilled water, stir and heat until gelatinization and transparency, let stand and cool for later use.
[0107] Aqueous phase preparation: Under stirring conditions, add 12.5 mL of an aqueous solution containing 0.3 g of STMP and 0.03 g of STPP to 12.5 mL of starch solution (mass ratio: soluble starch solution: corn amylopectin solution = 1:5), and stir for 5 minutes to mix thoroughly.
[0108] Preparation of microspheres: Under stirring conditions, add 1g of Span60 and 0.5g of Teween60 to 100mL of cyclohexane to form a continuous oil phase; after stirring for a period of time, add 25mL of water phase to the oil phase. After thorough mixing, stir, the reaction temperature is 70°C, after reacting for 3h, add 0.3g of STMP and 0.03g of STPP and continue to react for 21h, then stir overnight at room temperature, stir without standing during the whole process, add a certain amount of ethyl acetate, filter, and wash with ethyl acetate 3 times to remove oil-soluble impurities, rinse with purified water to obtain starch embolization microspheres.
[0109] (2) Testing the degradation behavior of starch embolization microspheres:
[0110] ① The starch embolic microspheres obtained in step (1) are precisely sieved to obtain starch embolic microspheres with a particle size of 100 to 300 μm, replaced with physiological saline three times, and centrifuged to remove excess solvent in the starch embolic microspheres to obtain starch embolic microspheres to be tested.
[0111] ② Take a 20mL vial and weigh it, measure 2mL of the starch embolic microspheres to be tested and transfer it into the vial, add ethanol and acetone for dehydration, and then vacuum dry it. After the vial cools to room temperature, weigh it, calculate the dry weight corresponding to 2mL of the starch embolic microspheres to be tested, and take the average mass of the three sets of parallel samples.
[0112] ③ Take 2 mL of starch embolization microspheres to be tested, and use the GBT 6913-2008 total phosphate content determination method and anthrone colorimetry to determine the sugar and phosphorus (in terms of PO4 3- Calculate the total mass of the solids (take this as the initial degradation value) and compare the difference between the initial degradation value and the average dry weight obtained in step ②.
[0113] ④ Measure 2mL of the starch embolization microspheres to be tested and add them to a 50mL centrifuge tube. Use Tris-HCL buffer with a pH of 7.4 to repeatedly rinse the measuring cylinder to ensure that there are no microspheres remaining on the wall of the measuring cylinder. Then add the simulated liquid to the 50mL centrifuge tube until the volume in the centrifuge tube is 50mL. Place the centrifuge tube in a water bath shaker at 37°C and 70rpm for incubation. During the incubation process, replace the liquid every 24h to remove the liquid phase in the centrifuge tube (the liquid phase does not contain the starch embolization microspheres to be tested, and the starch embolization microspheres to be tested are retained in the centrifuge tube), and then add the simulated liquid to the centrifuge tube until the volume in the centrifuge tube is 50mL.
[0114] The simulated liquid contains porcine pancreatic α-amylase and Tris-HCl buffer; the pH of the Tris-HCl buffer is 7.4; the enzyme activity concentration of the porcine pancreatic α-amylase in the simulated liquid is the same as the enzyme activity concentration of the α-amylase in rabbit plasma.
[0115] Preparation of Tris-HCl buffer: Mix 50 mL of 0.1 mol / L Tris base solution with 42 mL of 0.1 mol / L HCl, and add water to adjust the volume to 100 mL.
[0116] Preparation of the simulated solution: Mix 1 mg / mL porcine pancreatic α-amylase stock solution (prepared) with Tris-HCl buffer.
[0117] ⑤ During the incubation process of step ④, the supernatant after centrifugation of the incubation system at 0d, 1d, 3d, 5d and 7d (referred to as liquid A) and all the liquid phase removed from the centrifuge tube (referred to as liquid B) were taken, diluted, and the concentration of glucose in liquid A was determined by the total phosphate content determination method of GBT6913-2008 and the anthrone colorimetric method, respectively. e and phosphate (PO4 3- The concentration C b , the concentration of glucose in solution B is C s and phosphate (PO4 3- The concentration C a , and the degradation rate DR of the starch embolization microspheres to be tested at different incubation times was calculated according to the following formula.
[0118] The formula is as follows:
[0119] M e =C e ×X A ×V A
[0120] M b =C b ×X A ×V A
[0121] M s =C s ×X B ×V B
[0122] M a =C a ×X B ×V B
[0123] M 总 =M s +M e+M a +M b
[0124]
[0125] Where:
[0126] M e is the mass of glucose in solution A.
[0127] C e is the concentration of glucose after dilution of solution A.
[0128] X A is the dilution multiple of solution A.
[0129] V A is the volume of liquid A.
[0130] M b is the phosphate in liquid A (in PO4 3- The quality of the meter).
[0131] C b The phosphate (PO4 3- The concentration of the
[0132] M s is the mass of glucose in solution B.
[0133] C s is the concentration of glucose after dilution of solution B.
[0134] X B is the dilution multiple of solution B.
[0135] V B is the volume of liquid B.
[0136] M a Phosphate in solution B (PO4 3- The quality of the meter).
[0137] C a The phosphate (PO4 3- The concentration of the
[0138] M 总 The amount of glucose and phosphate (PO4) reduced in starch embolization microspheres 3- The sum of the masses of the items (measured).
[0139] M0' is the mass of glucose and phosphate (in terms of PO4 3- The sum of the masses of the items (measured).
[0140] Comparative Example 1
[0141] This comparative example provides a test method for simulating the degradation process of starch embolic microspheres in vivo. The difference between this comparative example and the embodiment is only that: steps ① and ⑤ in step (2) are different.
[0142] The step ① in step (2) of this comparative example is as follows:
[0143] The starch embolic microspheres obtained in step (1) are precisely sieved to obtain starch embolic microspheres with a particle size of 100 to 300 μm, replaced with physiological saline three times, and centrifuged to remove excess solvent in the starch embolic microspheres to obtain starch embolic microspheres with different cross-linking degrees. The starch embolic microspheres with low cross-linking degrees and the starch embolic microspheres with high cross-linking degrees are subjected to subsequent steps of simulating the degradation process in vivo.
[0144] Step ⑤ in step (2) of this comparative example is as follows:
[0145] During the incubation process of step ④, record the volume of the microspheres in the incubation system and the corresponding incubation time.
[0146] Comparative Example 2
[0147] This comparative example provides a test method for simulating the degradation process of starch embolic microspheres in vivo. The difference between this comparative example and the embodiment is only that: step ⑤ in step (2) is different; in this comparative example, step ⑤ in step (2) is as follows:
[0148] During the incubation process of step ④, the incubation system was centrifuged at incubation times of 0d, 1d, 3d, and 5d, and the supernatant after centrifugation was transferred into a vial (the weight of the vial corresponds to the weight of the bare vial in Table 4), and ethanol and acetone were added for dehydration and vacuum drying. After the vial cooled to room temperature, it was weighed (the weighing value corresponds to the value of starch embolism microspheres (dry weight) + vial in Table 4), and the dry weight of undegraded starch embolism microspheres was calculated (corresponding to the mass of the residue in Table 4). The mean mass of the three groups of parallel samples was compared with the mass of the sample with an incubation time of 0h, and the mass loss percentage of the starch embolism microspheres at each time point was calculated to evaluate the degradation process of the starch embolism microspheres in vivo.
[0149] Comparative Example 3
[0150] This comparative example provides a test method for simulating the degradation process of starch embolic microspheres in vivo. The difference between this comparative example and the embodiment is that the Tris-HCl buffer is replaced with physiological saline (pH 7.4).
[0151] Comparative Example 4
[0152] This comparative example provides a test method for simulating the degradation process of starch embolic microspheres in vivo. The difference between this comparative example and the embodiment is that porcine pancreatic α-amylase is replaced by Aspergillus oryzae α-amylase.
[0153] Comparative Example 5
[0154] This comparative example provides a test method for simulating the degradation process of starch embolic microspheres in vivo. The difference between this comparative example and the embodiment is that the simulation liquid is not replaced during the incubation process in this comparative example.
[0155] Experimental Example 1
[0156] The enzyme activity and shelf life of the simulated liquid of the embodiment were measured, and the results are shown in Table 1.
[0157] The simulated liquid of the embodiment was placed in a water bath shaker at 37° C. and 70 rpm for incubation, and samples were taken every day to measure the activity changes.
[0158] The 1 mg / mL porcine pancreatic α-amylase stock solution was stored at 4°C and samples were taken weekly to measure the activity changes.
[0159] Table 1
[0160]
[0161] As can be seen from Table 1, the α-amylase activity in rabbit plasma was approximately 2000 U / g, which served as a control.
[0162] As can be seen from Table 1, when the porcine pancreatic α-amylase mother solution is stored at 4°C, its stability can be maintained for 7 days; however, if the storage time of the porcine pancreatic α-amylase mother solution at 4°C is extended to 14 days, the enzymatic activity of the porcine pancreatic α-amylase will decrease significantly. Therefore, in order to ensure the accuracy of the experimental data, the prepared porcine pancreatic α-amylase mother solution needs to be used up within 7 days to avoid deviations in the experimental results due to reduced enzyme activity.
[0163] Furthermore, it can be seen from Table 1 that when the porcine pancreatic α-amylase was incubated at 37°C, the enzyme activity fluctuated slightly after 1 day of incubation, but after 2 days of incubation, the enzyme activity was reduced by half. This indicates that when using porcine pancreatic α-amylase for incubation degradation experiments, the simulated liquid containing porcine pancreatic α-amylase needs to be replaced regularly to supplement the simulated liquid containing fresh porcine pancreatic α-amylase, so as to maintain the activity of the enzyme and avoid the deviation of the experimental results caused by the reduction of enzyme activity.
[0164] Experimental Example 2
[0165] The degradation rates DR of the starch embolization microspheres (high cross-linking degree) to be tested at different incubation times in the examples were statistically analyzed, and the results are shown in Tables 2, 3 and Figure 2 As shown; and compared with the actual degradation process of the starch embolic microspheres to be tested in vivo.
[0166] Among them, the actual degradation process of the starch embolization microspheres to be tested in vivo was conducted using a rabbit ear embolization experiment.
[0167] The rabbit ear embolization experiment was as follows: New Zealand white rabbits (male, weighing 2.6-3.0 kg) were used for in vivo embolization studies. The hair on the back of the entire rabbit ear was removed before administration, and anesthesia was performed by intraperitoneal injection of Zotalar. The prepared starch embolic microspheres were mixed and suspended with iohexol and then slowly injected into the central artery of the rabbit ear (0.3-0.8 mL / ear). After the operation, the injection site was pressed for 30 seconds to stop bleeding. At 2h, 5d, and 7d after the operation, photos were taken to observe the changes in rabbit ear embolism and H&E stained sections were made to evaluate the embolization effect. Figures 3 to 5 The following are the results of rabbit ear embolization at 2 hours, 5 days and 7 days after surgery.
[0168] In step (2) ② of the example, the masses of the three parallel samples of the dry weight corresponding to 2 mL of the starch embolic microspheres (high cross-linking degree) to be tested were 138.8 mg, 147.8 mg and 125.7 mg respectively, and the average mass of the three parallel samples was 137.4 mg.
[0169] Table 2
[0170]
[0171]
[0172] In Table 1, "the sum of the mass of total sugar and total phosphorus in the dry weight of the starch embolization microspheres to be tested corresponding to different incubation times" is M0'-M 总 .
[0173] According to the data in Table 1, a normalized curve was drawn between the incubation time (i.e., degradation time) and the degradation rate to obtain the data in Tables 3 and Figure 4 .
[0174] Table 3
[0175]
[0176] from Figures 3 to 5 It can be seen that the starch embolic microspheres can be successfully embolized 2 hours after injection into the rabbit ear, and the starch embolic microspheres maintain their intact morphology. Five days after surgery, the color of the embolized area became lighter and the starch embolic microspheres were damaged, indicating that the starch embolic microspheres had been partially degraded. Seven days after surgery, the blood vessels at the embolized area of the rabbit ear were recanalized and the starch embolic microspheres were completely degraded.
[0177] Combined with Table 2, Table 3, Figures 2 to 5It can be seen that by monitoring the total phosphorus and total sugar content in the degraded starch embolic microspheres, the in vitro degradation trend of the starch embolic microspheres simulated in the embodiment is consistent with the in vivo degradation process. Specifically, the in vitro degradation experiment of the starch embolic microspheres simulated in the embodiment shows that the starch embolic microspheres have a degradation rate of 50% within 3 days and are completely degraded within 7 days, which is consistent with the 7-day vascular recanalization trend observed in the in vivo experiment.
[0178] By comparing the total phosphorus and total sugar results with the mass difference of the microspheres after drying, we found that there was little difference between the two, which verified the accuracy of the test method of simulating the degradation process of starch embolization microspheres in vivo in the embodiment.
[0179] The test data between the parallel groups were highly consistent, indicating that the test method for simulating the degradation process of starch embolization microspheres in vivo in the embodiment has high stability.
[0180] In addition, the degradation of the starch embolic microspheres (low cross-linking degree) to be tested at different incubation times in the embodiment is consistent with the 7-day vascular recanalization trend observed in the in vivo experiment, and the test data between parallel groups are consistent with each other, indicating that the test method of simulating the degradation process of starch embolic microspheres (low cross-linking degree) in vivo in the embodiment also has high accuracy and stability.
[0181] Experimental Example 3
[0182] The degradation process of starch embolic microspheres was analyzed by the test method for simulating the degradation process of starch embolic microspheres in vivo provided in Comparative Example 1. The results are as follows: Figure 6 and Figure 7 shown.
[0183] Figure 6 The degradation process of starch embolic microspheres with low cross-linking degree; Figure 6 In the figure, the volume corresponding to the black line in A is the volume of the starch embolic microspheres at 0 h of incubation, the volume corresponding to the black line in B is the volume of the starch embolic microspheres at 2 h of incubation, the volume corresponding to the black line in C is the volume of the starch embolic microspheres at 2 d of incubation, and the volume corresponding to the black line in D is the volume of the starch embolic microspheres at 2.92 d of incubation.
[0184] Figure 7 The degradation process of starch embolic microspheres with high cross-linking degree; Figure 7 In the figure, the volume corresponding to the black line in A is the volume of the starch embolic microspheres at 0 h of incubation, the volume corresponding to the black line in B is the volume of the starch embolic microspheres at 1 d of incubation, the volume corresponding to the black line in C is the volume of the starch embolic microspheres at 2 d of incubation, the volume corresponding to the black line in D is the volume of the starch embolic microspheres at 3 d of incubation, the volume corresponding to the black line in E is the volume of the starch embolic microspheres at 5 d of incubation, and the volume corresponding to the black line in F is the volume of the starch embolic microspheres at 7 d of incubation.
[0185] from Figure 6 and Figure 7 It can be seen that the volume of starch embolic microspheres with low cross-linking degree gradually decreases during the degradation process; while starch embolic microspheres with high cross-linking degree show a different degradation pattern, and their volume first increases and then decreases.
[0186] Therefore, the degradation trend of starch embolic microspheres in vivo is evaluated by monitoring the volume change of starch embolic microspheres. This method is only applicable to starch embolic microspheres with low cross-linking degree. For starch embolic microspheres with high cross-linking degree, the volume change may not accurately reflect the actual degradation of starch embolic microspheres in vivo. The test method for simulating the degradation process of starch embolic microspheres in vivo provided in Comparative Example 1 is not universal.
[0187] Experimental Example 4
[0188] The degradation process of starch embolic microspheres was analyzed by the test method for simulating the degradation process of starch embolic microspheres in vivo provided in Comparative Example 2. The results are shown in Table 4.
[0189] Table 4
[0190]
[0191] It can be seen from Table 4 that in the stage of low degradation rate, it is reasonable to reflect the consistency of the degradation trend of starch embolic microspheres with the in vivo degradation through quality monitoring. In this stage, the degradation trend of starch embolic microspheres was reflected by detecting the mass loss of degradation residues, which showed that the degradation behavior of starch embolic microspheres was consistent with the in vivo degradation.
[0192] However, as the degradation rate increases, when the degradation residue becomes very small, the mass loss during the drying process becomes significant, which results in the in vitro degradation results being unable to accurately reflect the actual situation of in vivo degradation. Therefore, the universality of the test method for simulating the degradation process of starch embolization microspheres in vivo provided in Comparative Example 2 is not high.
[0193] Experimental Example 5
[0194] The degradation time corresponding to the test methods for simulating the degradation process of starch embolization microspheres in vivo provided in Examples and Comparative Examples 3 to 5 were compared, and the results are shown in Table 5. The pH change of the simulated liquid in Comparative Example 5 with incubation time was tested, and the results are shown in Table 6.
[0195] Table 5
[0196]
[0197] Table 6
[0198]
[0199] It can be seen from Table 5 that only the time for the starch embolic microspheres to be completely degraded in vitro in Example and Comparative Example 5 is consistent with the actual degradation time in vivo in Experimental Example 2.
[0200] As can be seen from Table 6, when physiological saline is used as the degradation medium of the simulated liquid, the pH of the simulated liquid will change with the incubation time, which cannot simulate the real enzyme degradation environment in the body.
[0201] In summary, the present application uses a specific simulation liquid and replaces the simulation liquid at specific preset time intervals, so as to simulate the real enzymatic degradation environment of starch embolic microspheres in vivo; by obtaining the relationship between the reduced sugar mass in the starch embolic microspheres and the incubation time, it reflects the change of the degradation of starch embolic microspheres with the incubation time, and thus can more accurately simulate the real degradation process of starch embolic microspheres in vivo; the method provided in the present application can be used to evaluate the degradation behavior of starch embolic microspheres in vivo, which can provide a scientific basis for the registration and approval process of medical devices, and also help the development and optimization of starch embolic microsphere materials, reducing the risks and costs of clinical trials.
[0202] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.
Claims
1. A test method for simulating the degradation process of starch embolic microspheres in vivo, characterized in that: include: Placing the starch embolization microspheres in a simulated solution for incubation; and during the incubation process, removing the original simulated solution at a preset time interval and adding new simulated solution; Obtaining the relationship between the reduced sugar mass in the starch embolization microspheres and the incubation time to determine the degradation process of the starch embolization microspheres in vivo; Wherein, the simulated liquid contains porcine pancreatic α-amylase and Tris-HCl buffer; The pH of the Tris-HCl buffer is 7.35-7.45; the enzyme activity concentration of the porcine pancreatic α-amylase in the simulated solution is the same as the enzyme activity concentration of the α-amylase in the simulated plasma in vivo; The incubation temperature is 36-38°C; The preset time is ≤24h.
2. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 1, characterized in that: The preset time is 18 to 24 hours.
3. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 1, characterized in that: The incubation is carried out in a shaker with a rotation speed of 60 to 80 rpm.
4. The test method for simulating the degradation process of starch embolic microspheres in vivo according to any one of claims 1 to 3, characterized in that: According to formula (1), the reduced sugar mass M in the starch embolization microspheres is obtained: t ; Formula (1) is as follows: M t =M s +M e Where M s is: the total mass of all sugars in the simulated liquid removed during the incubation process; M e is: the mass of sugar in the supernatant after centrifugation of the incubated system.
5. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 4, characterized in that: The testing method comprises: obtaining the relationship between the degradation rate of the starch embolization microspheres and the incubation time according to the relationship between the reduced sugar mass in the starch embolization microspheres and the incubation time, so as to determine the degradation process of the starch embolization microspheres in vivo; Wherein, the degradation rate DR of the starch embolization microspheres is obtained according to formula (2); Formula (2) is as follows: Wherein, M0 is: the mass of sugar in the starch embolization microspheres before the incubation.
6. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 4, characterized in that: The sugar concentration in all the simulated fluids removed was tested to determine the M s ; Testing the sugar concentration in the supernatant to determine the M e ; Optionally, the concentration of sugar in all the removed simulated liquids and the concentration of sugar in the supernatant are tested by anthrone colorimetry.
7. The test method for simulating the degradation process of starch embolic microspheres in vivo according to any one of claims 1 to 3, characterized in that: Before the incubation, the starch embolization microspheres are pretreated; The pretreatment step includes: replacing the starch embolic microspheres with physiological saline, collecting the precipitate after centrifugation, and obtaining the starch embolic microspheres from the precipitate; Optionally, before the pretreatment, the particle size of the starch embolization microspheres is 100 to 300 μm.
8. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 7, characterized in that: placing a preset volume of the starch embolization microspheres in the simulated liquid for incubation; The volume ratio of the preset volume to the simulated liquid during the incubation process is 1:(20-30).
9. The test method for simulating the degradation process of starch embolic microspheres in vivo according to any one of claims 1 to 3, characterized in that: The starch embolic microspheres have a core-shell structure, and the degradation rate of the core material of the starch embolic microspheres is greater than the degradation rate of the shell material of the starch embolic microspheres; Optionally, the core material is a soluble starch polymer, or / and, the shell material is a pullulan polymer; Optionally, in the starch embolization microspheres, the mass ratio of the core material to the shell material is (1-5):(1-5).
10. The test method for simulating the degradation process of starch embolic microspheres in vivo according to claim 9, characterized in that: The preparation method of the starch embolization microspheres comprises: The microsphere skeleton material is formulated into an aqueous phase, wherein the microsphere skeleton material comprises the core material and the shell material, the hydrophilicity of the core material is greater than the hydrophilicity of the shell material; and the aqueous phase comprises sodium trimetaphosphate and sodium tripolyphosphate; The aqueous phase and the oil phase are mixed to form an inverse suspension polymerization system, and then a polymerization reaction is performed to obtain the starch embolization microspheres; The test method comprises: obtaining the relationship between the sum of the reduced sugar mass and phosphorus mass in the starch embolism microspheres and the incubation time to determine the degradation process of the starch embolism microspheres in vivo; Optionally, the sum of the reduced sugar mass and phosphorus mass in the starch embolization microspheres M is obtained according to formula (3): 总 ; Formula (3) is as follows: M 总 =M s +M e +M a +M b Where M s is: the total mass of all sugars in the simulated liquid removed during the incubation process; M e is: the mass of sugar in the supernatant after centrifugation of the incubated system; M a is: the total mass of phosphorus in all the simulated liquids removed during the incubation process; M b is: the mass of phosphorus in the supernatant after centrifugation of the incubated system.